Anthrax
The Agent
Anthrax is a serious disease that came into public prominence in 2001 during the bioterrorism attack in the United States. Anthrax is caused by a bacterium called Bacillus anthracis (B. anthracis). The name anthrax comes from the Greek word for coal and refers to the black skin lesions it produces. Descriptions of a disease affecting both animals and humans that appear to be anthrax have been found as early as Biblical times, and in fact anthrax has been suggested to have been the fifth plague described in the book of Exodus.
The anthrax bacterium was first described in 1823 and was the first bacterium ever shown to be the cause of a disease - in 1876, Robert Koch obtained a pure culture of B. anthracis and demonstrated that it caused disease by injecting it into animals. B. anthracis was also the first bacterium to be used for making an attenuated vaccine by Louis Pasteur in 1881.
Courtesy: CDC/
Janice Carr
B. anthracis is a large, rod-shaped bacterium that forms spores. These spores can survive in a dormant state in the environment, usually in soil, for many years, even decades. Once ingested, the spores are activated, and the bacteria begin to reproduce. Reproducing bacteria produce three different proteins that combine to form two toxins known as lethal toxin and edema toxin. The toxins cause a fatal buildup of fluid around the lungs that can kill infected cells and produce disease and death in infected animals or humans.
Anthrax is primarily a disease of livestock that become infected by ingesting spores found in soil. Humans usually become infected with anthrax by handling products of infected animals such as leather or wool or by inhaling anthrax spores from infected animal products. They can also become infected by eating undercooked meat from infected animals. Anthrax is not known to be spread person-to-person. Cases of transmission of anthrax from infected animals to humans are relatively rare in the United States, with an average of about five cases per year. However, in 2001, there were 22 cases of anthrax infection that were caused by deliberate spread through the United States Postal system. Letters containing anthrax killed five people and sickened 17 others and caused a temporary disruption of mail service and the forced evacuation of several buildings including Senate offices and the Supreme Court. After a massive and difficult seven year investigation, the Federal Bureau of Investigation concluded this case after its leading and sole suspect, an Army microbiologist, committed suicide.
There are three main forms of human anthrax.
- Cutaneous anthrax occurs when the bacteria from infected animal products enter a break in the skin; black lesions occur at the site of infection. This is the most common form of anthrax and can be controlled with antibiotics if it is treated before the infection spreads through the body.
- Gastrointestinal anthrax can occur from the ingestion of contaminated food and can be fatal if not treated immediately. This form is not known to have occurred in the United States.
- Inhalation anthrax occurs when anthrax spores are inhaled. The spores travel to the lymph nodes near the lungs and produce toxins that cause severe breathing problems and shock. This form is very difficult to treat and is often lethal. Naturally occurring inhalation anthrax is very rare, but this was the type of anthrax infection that occurred during the bioterrorism attack of 2001. This is the most dangerous form, and half of the inhalation cases of anthrax led to death during the 2001 attack.
The Problem
The bacterium that causes anthrax is considered a highly dangerous potential agent for use in bioterrorism and is classified as a Category A agent by the Centers for Disease Control and Prevention ( CDC) – the highest risk type of agent.
The reasons that anthrax is so dangerous are that
- it is highly toxic – the mortality rate is nearly 100% for the inhalation form in the absence of treatment.
- its spores are easily disseminated through the air.
- the spores are extremely durable.
Although several different antibiotics exist that are effective against anthrax, the early symptoms are often confused with respiratory or gastrointestinal diseases, and once the obvious symptoms occur, it is usually too late to counteract the destructive effects of the anthrax toxins. There is also the serious concern that the anthrax bacterium could become resistant to currently used antibiotics, and in fact some strains are already resistant to certain classes of antibiotics.
There is currently a vaccine against the bacterium that causes anthrax, but its use is restricted to military personnel and workers with an occupational risk for anthrax exposure. Its safety or effectiveness in children, the elderly, and people with weakened immune systems has not been determined. The major problem with the current vaccine, however, is that it is given as six doses over an 18-month period, so that it is unlikely to provide much protection to the general public in the event of a widespread terrorist attack.
The Research
In the Department of Molecular Virology and Microbiology (MVM) at Baylor College of Medicine (BCM), research on anthrax is being performed in several areas – vaccine development, antibiotic resistance, the identification and analysis of all of the proteins that make up the anthrax bacterium (an approach called proteomics), and an investigation into the mechanisms by which B. anthracis acquires the essential nutrient iron and overcomes a normal host defense against infection.
The anthrax bacterium produces several toxins that are responsible for many of the disease manifestations. Antibodies to one part of the toxins - the protective antigen (PA) have been found to protect against infection and disease. Therefore, most new vaccines for protection against anthrax consist of or contain the PA protein. Development of new vaccines and of new ways of delivering the licensed vaccine is a major focus of the BCM Vaccine and Treatment Evaluation Unit (VTEU) headed by Dr. Wendy Keitel. This effort involves the evaluation of a diverse group of licensed and experimental products.
Several clinical trials to evaluate anthrax vaccines are in progress.
- Anthrax vaccine adsorbed (AVA) is the only vaccine licensed for use in the United States. Immunization with AVA requires frequent doses of vaccine given subcutaneously (or under the skin), and commonly is associated with reactions at the site of injection. MVM researchers are currently conducting a CDC-sponsored phase II clinical trial of AVA. The goals of this study are to develop a simpler and better-tolerated way of giving the vaccine, and to provide information that will contribute to the development of a new anthrax vaccine. Healthy volunteers are given the standard number of injections under the skin or into the muscle, or fewer doses into the muscle. Reactions and immune responses after vaccination will be compared between the different groups. Antibody and cell-mediated immune responses will be measured and correlated with responses that are associated with protection in animal models.
- Another study is a VTEU-supported phase I clinical trial of a candidate DNA vaccine consisting of two segments of DNA expressing PA and lethal factor, two of the three components of anthrax toxins. The goals of this study are to evaluate the safety of the vaccine, and to determine whether the vaccine can stimulate antibody responses that are associated with protection against infection (i.e., to test the immunogenicity of the vaccine).
- A third clinical study is a phase I evaluation of a purified recombinant PA protein vaccine, sponsored by VaxGen, Inc. through an accelerated program funded by NIAID (a component of the National Institutes of Health). The goals of this study are to evaluate the safety and immunogenicity of the vaccine, and to select dosage levels for evaluation in larger phase II clinical trial.
Drs. Keitel, Hanaa El Sahly and colleagues have recently reported results from the first of these clinical trials in which they sought to simplify the vaccination schedule and reduce the side effects. The researchers evaluated the antibody response and side effects in groups of healthy adults who were given three doses of the anthrax vaccine compared to the usual four doses and who received the injection in the muscle rather than under the skin. They found that giving fewer injections into the muscle was comparable to the usual regimen of four doses given under the skin. They also observed fewer and milder side effects in individuals who received the injections into the muscle. Reducing the number of times the vaccine must be given, and the adverse effects, will reduce the cost and make it easier to fully vaccinate people against anthrax.
Another group of MVM researchers is studying antibiotic resistance of the anthrax bacterium. Treatment for anthrax infections includes use of the antibiotics ciprofloxacin, doxycycline, and penicillin G. The problem is that some strains of the anthrax bacterium are naturally resistant to penicillin, so that penicillin is ineffective in treating patients with these particular strains. Resistance to penicillin is often due to enzymes made by bacteria that are called β-lactamases (these enzymes basically break apart penicillin). One strain of B. anthracis makes two such enzymes (called Bla1 and Bla2). Dr. Timothy Palzkill and his group are studying these enzymes to learn in detail how they allow some strains of B. anthracis to resist antibiotics. Their goal is to develop compounds that can inhibit the action of the β-lactamase enzymes, so that they are no longer effective in blocking the action of antibiotics.
A third area of anthrax research utilizes an approach called functional genomics. Drs. Joseph Petrosino, George Weinstock, and Timothy Palzkill are studying the genome sequences of different B. anthracis strains and using this information to understand critical areas of B. anthracis biology. The genome sequence of multiple B. anthracis strains and other Bacillus species have been determined. Two independent virulence plasmids distinguish B. anthracis from other Bacillus species. These plasmids, required for B. anthracis virulence, encode 224 genes, including the three anthrax toxin subunits (Protective Antigen-PA, Lethal Factor-LF, and Edema Factor-EF). Because of their uniqueness to B. anthracis, these virulence plasmid genes have been targeted for further study.
Drs. Petrosino and Weinstock have cloned each of the 224 virulence plasmid genes for the purpose of expression and purification in laboratory strains of Escherichia coli. Purified proteins are being used in immunological screens with immune sera from Rhesus macaque monkeys that have been infected with B. anthracis (in collaboration with Dr. Conrad Quinn, CDC and Dr. Johnny Peterson, UTMB). The screens are designed to identify virulence plasmid-encoded proteins recognized by the macaque immune response to anthrax. Subsequent screens will test sera from human volunteers immunized with the current anthrax vaccine to determine which virulence plasmid-encoded proteins are present in the vaccine and are recognized by the human immune response. The proteins identified will be unique to anthrax and may be ideal candidates for further study in the development of a new generation of anthrax subunit vaccines and diagnostic tools.
Studies by Dr. Anthony Maresso investigate iron acquisition by B. anthracis. Iron is an essential nutrient used by almost all organisms. Bacterial pathogens must acquire iron in order to grow inside mammalian hosts. The host, however, limits the availability of free iron, thereby providing an effective defense strategy against infection. In response, bacteria have evolved clever ways to subvert host sequestration of iron. Dr. Maresso has uncovered secreted proteins produced by B. anthracis which specifically bind and transport the body’s prominent iron-carrier molecule, heme. The acquisition of mammalian heme may allow B. anthracis to attain enough iron to grow to very high densities during infection. Dr. Maresso has also uncovered a group of small molecule inhibitors which specifically target and inactivate enzymes in the pathway of iron acquisition in pathogens like B. anthracis. An understanding of the mechanisms of iron uptake in B. anthracis will allow for the development of therapeutic agents to combat infections by related Gram-positive bacteria.
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